Epigenetic changes induced by pathogenic Chlamydia spp.

Chlamydia trachomatis, C. pneumoniae, and C. psittaci, the three Chlamydia species known to cause human disease, have been collectively linked to several pathologies, including conjunctivitis, trachoma, respiratory disease, acute and chronic urogenital infections and their complications, and psittacosis. In vitro, animal, and human studies also established additional correlations, such as between C. pneumoniae and atherosclerosis and between C. trachomatis and ovarian cancer. As part of their survival and pathogenesis strategies as obligate intracellular bacteria, Chlamydia spp. modulate all three major types of epigenetic changes, which include deoxyribonucleic acid (DNA) methylation, histone post-translational modifications, and microRNA-mediated gene silencing. Some of these epigenetic changes may be implicated in key aspects of pathogenesis, such as the ability of the Chlamydia spp. to induce epithelial-to-mesenchymal transition, interfere with DNA damage repair, suppress cholesterol efflux from infected macrophages, act as a co-factor in human papillomavirus (HPV)-mediated cervical cancer, prevent apoptosis, and preserve the integrity of mitochondrial networks in infected host cells. A better understanding of the individual and collective contribution of epigenetic changes to pathogenesis will enhance our knowledge about the biology of Chlamydia spp. and facilitate the development of novel therapies and biomarkers. Pathogenic Chlamydia spp. contribute to epigenetically-mediated gene expression changes in host cells by multiple mechanisms.

TLR2 mediates autophagy through ERK signaling pathway in Chlamydia psittaci CPSIT_p7 protein-stimulated RAW264.7 cells.

Chlamydia psittaci is a zoonotic pathogen found in birds and humans. Macrophages, major components of the innate immune system, can resist chlamydial infections and trigger adaptive immune responses. However, the molecular mechanisms underlying the action of macrophages against C. psittaci infection are not well understood. This study investigated the roles and mechanisms of plasmid-encoded protein CPSIT_p7 of C. psittaci in regulating autophagy in RAW264.7 cells. The results demonstrated that stimulation of RAW264.7 with C. psittaci plasmid protein CPSIT_p7 induced the expressions of the autophagy signaling primary regulators LC3 and Beclin1, which could also significantly induce the phosphorylation levels of ERK, JNK, p38, and Akt. Next, siRNA knockdown of TLR2 resulted in significant downregulation of CPSIT_p7-triggered autophagy in RAW264.7 cells. Moreover, the extracellular regulated protein kinase (ERK) inhibitor PD98059 markedly reduced autophagy in CPSIT_p7-stimulated macrophages. In summary, these results indicated that TLR2 plays an essential role in the induction of autophagy through the ERK signaling pathway in CPSIT_p7-stimulated RAW264.7 cells.

MOMP and MIP DNA-loaded bacterial ghosts reduce the severity of lung lesions in mice after Chlamydia psittaci respiratory tract infection.

Chlamydia psittaciis a well known zoonotic pathogen that can lead to severe respiratory disease in poultry, pet birds and humans. Development of an effective and safe vaccine would be the most effective way to control C. psittaci infection. In this study, we used bacterial ghosts (BGs) as a delivery vehicle to evaluate the protective effects of major outer membrane protein (MOMP) and macrophage infectivity potentiator (MIP) DNA vaccines in mice. We found that MOMP/MIP DNA-loaded BGs elicited a better immune response than a naked DNA vaccine, giving increased IgG titers, lymphocyte proliferation responses and higher levels of IFN-γ. After challenge infection, MOMP/MIP DNA-loaded BGs-immunized mice showed lower chlamydial load and inflammation pathology in lung tissues. In addition, we found that MOMP and MIP co-immunization or a heterologous prime-boost strategy could induce stronger immune responses and better protective efficacy against C. psittaci infection. Together, the above results suggest that BGs can act as an effective delivery vehicle for C. psittaci DNA vaccines, and co-immunization or heterologous prime-boost strategy can enhance protective efficacy against infection, thereby providing an alternative strategy for the design of vaccines against C. psittaci.

Localization and characterization of a putative cysteine desulfurase in Chlamydia psittaci.

Chlamydia psittaci is an obligate intracellular pathogen with a biphasic developmental life cycle. It is auxotrophic for a variety of essential metabolites and obtains amino acids from eukaryotic host cells. Chlamydia can develop inside host cells within chlamydial inclusions. A pathway secreting proteins from inclusions into the host cellular cytoplasm is the type III secretion system (T3SS). The T3SS is universal among several Gram-negative bacteria. Here, we show that CPSIT_0959 of C. psittaci is expressed midcycle and secreted into the infected cellular cytoplasm via the T3SS. Recombinant CPSIT_0959 possesses cysteine desulfurase and PLP-binding activity, which removes sulfur from cysteine to produce alanine, and helps chlamydial replication. Our study shows that CPSIT_0959 improve the infectivity of offspring elementary bodies and seems to promote the replication by its product. This phenomenon has inhibited by the PLP-dependent enzymes inhibitor. Moreover, CPSIT_0959 increased expression of Bim and tBid, and decreased the mitochondrial membrane potential of host mitochondria to induce apoptosis in the latecycle for release of offspring. These results demonstrate that CPSIT_0959 has cysteine desulfurase and PLP-binding activity and is likely to contribute to apoptosis of the infected cells via a mitochondria-mediated pathway to improve the infectivity of progeny.

Epidemiological and molecular characteristics of Chlamydia psittaci from 8 human cases of psittacosis and 4 related birds in Argentina / Características epidemiológicas y moleculares de Chlamydia psittaci provenientes de 8 casos humanos de psitacosis y de 4 aves relacionadas en la Argentina

In Argentina, the epidemiological and molecular characteristics of Chlamydia psittaci infections are still not sufficiently known. A total of 846 respiratory and 10 ocular samples from patients with suspected human psittacosis were tested for C. psittaci from January 2010 to March 2015. Four samples of birds related to these patients were also studied. Forty-eight samples were positive for C. psittaci by a nested PCR. The molecular characterization of twelve C. psittaci PCR-positive samples received in the National Reference Laboratory INEI-ANLIS "Dr. Carlos G. Malbrán", Buenos Aires, Argentina was performed. Eight positive samples from humans and four from birds were genotyped by ompA gene sequencing. C. psittaci genotype A was found in all human samples and in the related birds. This report contributes to our increasing knowledge of the epidemiological and molecular characteristics of C. psittaci to conduct effective surveillance of its zoonotic infections.

En la Argentina, aún no se conocen suficientemente las características epidemiológicas y moleculares de las infecciones por Chlamydia psittaci. Entre enero del 2010 y marzo del 2015 se estudiaron 846 muestras respiratorias y 10 oculares de pacientes con sospecha de psitacosis para la búsqueda de C. psittaci. También se estudiaron 4 muestras de aves relacionadas con estos pacientes. De ese total, 48 muestras fueron positivas para C. psittaci mediante una reacción en cadena de la polimerasa (PCR) anidada. Posteriormente, se realizó en el INEI-ANLIS «Dr. Carlos G. Malbrán¼ la caracterización molecular de 12 muestras positivas para C. psittaci, 8 de humanos y 4 de aves, que fueron genotipificadas por secuenciación del gen ompA. C. psittaci genotipo A se encontró en todas esas muestras. Este informe contribuye a mejorar nuestro conocimiento de las características epidemiológicas y moleculares de C. psittaci para lograr una vigilancia efectiva de la zoonosis que produce.

The JAK/STAT3 signaling pathway mediates inhibition of host cell apoptosis by Chlamydia psittaci infection.

The JAK-STAT3 signaling pathway is a key regulator of cell growth, motility, migration, invasion and apoptosis in mammalian cells. Infection with intracellular pathogens of the genus Chlamydia can inhibit host cell apoptosis, and here we asked whether the JAK-STAT3 pathway participates in chlamydial anti-apoptotic activity. We found that, compared with uninfected cells, levels of JAK1 and STAT3 mRNA as well as total and phosphorylated JAK1 and STAT3 protein, were significantly increased in C. psittaci-infected HeLa cells. Moreover, the apoptosis rate of infected cells was higher after treatment with the tyrosine kinase inhibitor AG-490 (2-cyano-3-(3, 4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide). Immunoblotting of apoptosis-related proteins showed that C. psittaci infection reduces Bax, but increases Bcl-2, protein levels, resulting in reduced activation of caspase-3, caspase-7, caspase-9 and PARP; AG490 attenuates these effects. Together, our data suggest that the JAK/STAT3 signaling pathway facilitates the anti-apoptotic effect of C. psittaci infection by reducing the Bax/Bcl-2 apoptotic switch ratio, and by inhibiting the intracellular activation of key pro-apoptotic enzymes.

Epidemiological and molecular characteristics of Chlamydia psittaci from 8 human cases of psittacosis and 4 related birds in Argentina.

In Argentina, the epidemiological and molecular characteristics of Chlamydia psittaci infections are still not sufficiently known. A total of 846 respiratory and 10 ocular samples from patients with suspected human psittacosis were tested for C. psittaci from January 2010 to March 2015. Four samples of birds related to these patients were also studied. Forty-eight samples were positive for C. psittaci by a nested PCR. The molecular characterization of twelve C. psittaci PCR-positive samples received in the National Reference Laboratory INEI-ANLIS "Dr. Carlos G. Malbrán", Buenos Aires, Argentina was performed. Eight positive samples from humans and four from birds were genotyped by ompA gene sequencing. C. psittaci genotype A was found in all human samples and in the related birds. This report contributes to our increasing knowledge of the epidemiological and molecular characteristics of C. psittaci to conduct effective surveillance of its zoonotic infections.

Examination of the in vivo immune response elicited by Chlamydia psittaci in chickens.

It has since long been reported that Chlamydia psittaci is endemic in the poultry industry in Belgium as well as in other European Countries. This can lead to major economic losses because of a lowered egg production, higher mortality and carcass condemnation. Nowadays, expensive antibiotic treatments are necessary to reduce mortality rate but this can lead to antibiotic resistance. Moreover, C. psittaci can easily be transmitted from birds to humans through the inhalation of pathogen-containing aerosols derived from feces and eye and nostril secretions. Therefore, the need for an efficient vaccine against C. psittaci is augmenting. However, more research is needed to develop such a vaccine. Knowledge on the immune mechanisms of C. psittaci infections is crucial to understand the pathogenesis of, and immunity to this zoonotic pathogen and to act as a basis for vaccination studies. This study has investigated the in vivo immune response evoked by C. psittaci in his natural host, the chicken. Excretion of C. psittaci, chlamydial antibody detection in sera, blood immune cells and the mRNA expression levels of different cytokines, chemokines and one Toll-like receptor were investigated in different organs (conchae, lungs, airsacs, harderian gland, bursa fabricius and spleen) at different time points post infection (6 h, 24 h, 48 h, 4 d, 6d, 8 d, 10 d, 14 d and 21 d). A higher frequency of cytotoxic CD8(+) T cells and monocytes/macrophages expressing the MHC II molecule were observed in the infected group. Several cytokines and chemokines are significantly upregulated during infection but remarkably also significantly downregulated, especially at late time points. Furthermore, the only Toll-like receptor investigated, TLR4, was also significant upregulated in several organs. This study can contribute on the elucidation on how C. psittaci interact with his host, leading to the developing of targets for effective vaccination and therapeutic strategies for infection.

The type III secretion system (T3SS) of Chlamydophila psittaci is involved in the host inflammatory response by activating the JNK/ERK signaling pathway.

Chlamydophila psittaci (C. psittaci) is a human zoonotic pathogen, which could result in severe respiratory disease. In the present study, we investigated the role and mechanism of the type III secretion system (T3SS) of C. psittaci in regulating the inflammatory response in host cells. C. psittaci-infected THP-1 cells were incubated with the specific T3SS inhibitor INP0007, inhibitors of ERK, p38, or JNK, and the levels of inflammatory cytokines were analyzed using Q-PCR and ELISA. The levels of ERK, p38, and JNK phosphorylation were analyzed by Western blot. Our results verified that INP0007 inhibited chlamydial growth in vitro, but the coaddition of exogenous iron completely reversed the growth deficit. INP0007 inhibited the growth of C. psittaci and decreased the levels of IL-8, IL-6, TNF-α, and IL-1ß. Exogenous iron restored the chlamydial growth but not the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3SS which reduced by incubation with ERK and JNK inhibitors, but not with p38 inhibitor. We concluded that the T3SS elicited inflammatory responses by activating the JNK or ERK signaling pathways in the infection of C. psittaci.

Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the response to doxycycline treatment in ocular adnexal lymphoma.

PURPOSE: To compare genome-wide DNA methylation profiles according to Chlamydophila psittaci (Cp) infection status and the response to doxycycline treatment in Korean patients with ocular adnexal extranodal marginal zone B-cell lymphoma (EMZL). METHODS: Twelve ocular adnexal EMZL cases were classified into two groups (six Cp-positive cases and six Cp-negative cases). Among the 12 cases, eight were treated with doxycycline as first-line therapy, and they were divided into two groups according to their response to the treatment (four doxy-responders and four doxy-nonresponders). The differences in the DNA methylation states of 27,578 methylation sites in 14,000 genes were evaluated using Illumina bead assay technology. We also validated the top-ranking differentially methylated genes (DMGs) with bisulfite direct sequencing or pyrosequencing. RESULTS: The Infinium methylation chip assay revealed 180 DMGs in the Cp-positive group (74 hypermethylated genes and 106 hypomethylated genes) compared to the Cp-negative group. Among the 180 DMGs, DUSP22, which had two significantly hypomethylated loci, was validated, and the correlation was significant for one CpG site (Spearman coefficient=0.6478, p=0.0262). Regarding the response to doxycycline treatment, a total of 778 DMGs were revealed (389 hypermethylated genes and 336 hypomethylated genes in the doxy-responder group). In a subsequent replication study for representative hypomethylated (IRAK1) and hypermethylated (CXCL6) genes, the correlation between the bead chip analysis and pyrosequencing was significant (Spearman coefficient=0.8961 and 0.7619, respectively, p<0.05). CONCLUSIONS: Ocular adnexal EMZL showed distinct methylation patterns according to Cp infection and the response to doxycycline treatment in this genome-wide methylation study. Among the candidate genes, DUSP22 has a methylation status that was likely attributable to Cp infection. Our data also suggest that the methylation statuses of IRAK1 and CXCL6 may reflect the response to doxycycline treatment.

A new role of the complement system: C3 provides protection in a mouse model of lung infection with intracellular Chlamydia psittaci.

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3(-/-) mice succumbing to infection after day 9. At a low infective dose, C3(-/-) mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3(-/-) mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.

Chlamydia psittaci-negative ocular adnexal marginal zone B-cell lymphomas have biased VH4-34 immunoglobulin gene expression and proliferate in a distinct inflammatory environment.

Ocular adnexal marginal zone B-cell lymphomas (OAMZLs) arise in the connective tissues of the orbit or in the mucosa-associated lymphoid tissue of the conjunctiva. Here, we present the immunological and genetic analyses of 20 primary Chlamydia psittaci (Cp)-negative OAMZLs. Analysis of the immunoglobulin variable heavy chain (IgV(H)) gene usage demonstrated a significant preference for V(H)4-34. A combined analysis across all previously published OAMZLs confirmed that this is a general feature of OAMZL, in particular of the Cp-negative group. Our series of OAMZLs did not express the characteristic rheumatoid factor V(H)DJ(H) rearrangements that were previously found in salivary gland- and gastric-marginal zone B-cell lymphomas (MZBCLs). We did not detect the MZBCL-specific chromosomal translocations, t(11;18) API2-MALT1 (mucosa-associated lymphoid tissue1) and t(14;18) IgH/MALT1. Two cases contained a premature stop codon in the A20 gene (TNFAIP3) and one case harbored the activating MYD88 hotspot mutation L265P. Variable nuclear expression of BCL10, NFκB1 (p50) and NFκB2 (p52) suggests that other additional genetic abnormalities affecting the NFκB pathway exist within this group of lymphomas. OAMZL showed variable expression of the chemokine receptor CXCR3 and integrin α4ß7 by the tumor B cells, and low interferon-γ and interlukin-4 mRNA levels in the tissue, indicative of an inflammatory environment with features in between those previously found in cutaneous and other extranodal MZBCL. The strongly biased usage of V(H)4-34 in Cp-negative OAMZLs suggests involvement of a particular stimulatory (auto-) antigen in their development.

Host genetics and Chlamydia disease: prediction and validation of disease severity mechanisms.

Genetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention. We have previously demonstrated that polymorphisms in immunity-related GTPases (IRG) confer a significant difference in susceptibility to Chlamydia psittaci infection in BXD recombinant mice. Here we combine genetic mapping and network modeling to identify causal pathways underlying this association. We infected a large panel of BXD strains with C. psittaci and assessed host genotype, IRG protein polymorphisms, pathogen load, expression of 32 cytokines, inflammatory cell populations, and weight change. Proinflammatory cytokines correlated with each other and were controlled by a novel genetic locus on chromosome 1, but did not affect disease status, as quantified by weight change 6 days after infection In contrast, weight change correlated strongly with levels of inflammatory cell populations and pathogen load that were controlled by an IRG encoding genetic locus (Ctrq3) on chromosome 11. These data provided content to generate a predictive model of infection using a Bayesian framework incorporating genotypes, immune system parameters, and weight change as a measure of disease severity. Two predictions derived from the model were tested and confirmed in a second round of experiments. First, strains with the susceptible IRG haplotype lost weight as a function of pathogen load whereas strains with the resistant haplotype were almost completely unaffected over a very wide range of pathogen load. Second, we predicted that macrophage activation by Ctrq3 would be central in conferring pathogen tolerance. We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden. Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility.

Differential cytokine expression in Chlamydophila psittaci genotype A-, B- or D-infected chicken macrophages after exposure to Escherichia coli O2:K1 LPS.

Chlamydophila (Cp.) psittaci and avian pathogenic Escherichia (E.) coli infections contribute to the respiratory disease complex observed in turkeys. Secondary infection with E. coli exacerbates Cp. psittaci pathogenicity and augments E. coli excretion. The innate immune response initiated by both pathogens in their avian host is unknown. We therefore determined the cytokine responses following Cp. psittaci infection and E. coli superinfection of avian monocytes/macrophages by examining gene transcripts of IL-1beta, IL-6, CXCLi2 (IL-8), CXCLi1 (K60), IL-10, IL-12alpha/beta, IL-18, TGF-beta4 and CCLi2 at 4h post-inoculation with different Cp. psittaci strains or 4h post-treatment with avian E. coli LPS of Cp. psittaci pre-infected HD11 cells. Cp. psittaci strains used were 84/55 and 92/1293 (highly virulent), CP3 (low virulent) and 84/2334 (phylogenetically intermediate between Cp. psittaci and Chlamydophila abortus). At 4h post chlamydial infection, an increased expression of IL-1beta and IL-6 as well as CXCLi2, CXCLi1 and CCLi2 was observed compared to levels in uninfected HD11 controls. This effect was less pronounced for the milder CP3 strain. The pro-inflammatory response of Cp. psittaci infected cells to E. coli LPS was significantly lowered compared to uninfected controls, especially when the cells were pre-infected with highly virulent Cp. psittaci strains. In both experiments, exceptionally high IL-10 and no TGF-beta4 responses were observed, and we propose that this could induce macrophage deactivation and NF-kappaB suppression. Consequently, pro-inflammatory and Th1-promoting responses to both the primary Cp. psittaci infection and E. coli would be inhibited, thus explaining the observed aggravated in vivo pathology.

[Two familial cases of psittacosis].

We report here 2 cases of psittacosis in a family. In the first case, a 51-year-old woman was admitted with fever, dry cough, and a chest radiograph showed increased opacity in the right upper lung field. On a diagnosis of atypical pneumonia, minocycline was given and her clinical symptoms and abnormal laboratory data were improved. The second case was her husband, a 58-year-old man who presented with fever 4 days after his wife's admission. His chest radiograph revealed increased opacity in the left lower lung field. The administration of azithromycin for 3 days attenuated his clinical symptoms and his abnormal laboratory data improved. The serum titer of complement fixation (CF) test and ELISA test against Chlamydophila psittaci were elevated in both cases on analysis of paired acute- and convalescent-phase serum speciments. The antigen of Chlamydophila was revealed from these parrots, which had been raised in their family. Therefore, we concluded that the psittacosis had originated from the parrots.

Evaluation of the polymerase chain reaction in comparison with other diagnostic methods for the detection of Chlamydia psittaci.

Various diagnostic methods exist for the detection of Chlamydia psittaci. In the current study, the test performance of polymerase chain reaction (PCR) was compared with other testing methods used in the diagnosis of C. psittaci. Tissue and fecal specimens (n = 119) of avian and mammalian origin were tested by PCR and one or more of the following methods: cell culture, enzyme-linked immunosorbent assay, and direct fluorescein-conjugated monoclonal antibody staining. Several gold standards, based on results of testing methods other than PCR, were used to calculate the following test performance characteristics of PCR: sensitivity and specificity, with their 95% confidence intervals; kappa statistics, a measure of intertest agreement; and lambda statistics, a chance-corrected estimate of the sensitivity and specificity. Overall, the test performance characteristics of PCR were low compared with the other testing methods. Possible reasons for the poor test performance of PCR in the current study include destruction of the organisms during storage, interference with the PCR by other reagents, or technical errors.

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

Role of H-2 and non-H-2-related genes in mouse susceptibility to Chlamydia psittaci.

The genetic control of mouse susceptibility to Chlamydia psittaci was investigated after intravenous inoculation of 2 x 10(5) plaque-forming units (pfu). Splenic counts 6 days after inoculation gave the level of susceptibility. Results from inoculation of mice from H-2 congenic strains with three different genetic backgrounds (B10, BALB and C3H) suggested that both the non-H-2 genes and the genes of H-2 complex or those closely associated with it, were responsible for the observed differences in the innate capacity of various inbred lines of mice to control bacterial load in their infected organs following a challenge with C. psittaci.

Family outbreaks of psittacosis in Israel.

Eight family outbreaks of clinical or subclinical psittacosis in Israel after exposure to infected birds were studied. Throat cultures for Chlamydia psittaci and serological tests for Chlamydia species, including strain TWAR, were obtained from 37 people. Cloacal smears and cultures of internal organs for C psittaci were taken from 9 dead birds. 62% of the people studied had symptoms, and 67% of the birds that died had previously been sick. Evidence for acute C psittaci infection was found in 81% of patients (30/37). Diagnosis was established in 22 by isolation of the causal organism from throat cultures and in 8 by positive IgM serology (reciprocal titre greater than or equal to 8) with evidence of acute seroconversion or clinical findings compatible with the disease, or both. No serological evidence for acute TWAR infection was found. All birds studied had microbiological evidence of C psittaci infection, and most had abnormal findings on necropsy.